RESUMO
Autoimmune thyroid diseases (AITD) such as Graves' disease (GD) or Hashimoto's thyroiditis (HT) are organ-specific diseases that involve complex interactions between distinct components of thyroid tissue. Here, we use spatial transcriptomics to explore the molecular architecture, heterogeneity and location of different cells present in the thyroid tissue, including thyroid follicular cells (TFCs), stromal cells such as fibroblasts, endothelial cells, and thyroid infiltrating lymphocytes. We identify damaged antigen-presenting TFCs with upregulated CD74 and MIF expression in thyroid samples from AITD patients. Furthermore, we discern two main fibroblast subpopulations in the connective tissue including ADIRF+ myofibroblasts, mainly enriched in GD, and inflammatory fibroblasts, enriched in HT patients. We also demonstrate an increase of fenestrated PLVAP+ vessels in AITD, especially in GD. Our data unveil stromal and thyroid epithelial cell subpopulations that could play a role in the pathogenesis of AITD.
Assuntos
Antígenos de Diferenciação de Linfócitos B , Doença de Graves , Doença de Hashimoto , Glândula Tireoide , Humanos , Doença de Graves/patologia , Doença de Graves/imunologia , Doença de Graves/genética , Doença de Graves/metabolismo , Glândula Tireoide/patologia , Glândula Tireoide/metabolismo , Doença de Hashimoto/patologia , Doença de Hashimoto/imunologia , Doença de Hashimoto/metabolismo , Doença de Hashimoto/genética , Antígenos de Diferenciação de Linfócitos B/metabolismo , Antígenos de Diferenciação de Linfócitos B/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Células Epiteliais da Tireoide/metabolismo , Células Epiteliais da Tireoide/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Transcriptoma , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Células Estromais/metabolismo , Células Estromais/patologia , Feminino , Fatores Inibidores da Migração de Macrófagos , Oxirredutases IntramolecularesRESUMO
PURPOSE: Peripheral helper T (Tph) cells have an important role in the induction of humoral immune responses and autoantibody production. Accordingly, it is feasible that this lymphocyte subset has a relevant role in the pathogenesis of autoimmune thyroid diseases (AITD). In this study we aim to analyze the levels and function of Tph cells in blood samples from patients with AITD. METHODS: We performed an observational study with cases and controls. Blood samples were obtained from nineteen patients with Hashimoto's thyroiditis (HT), twenty-four with Graves' disease (GD), and fifteen healthy controls. In addition, the levels of follicular T helper (Tfh) cells and Tph cells, the release of interleukin-21 (IL-21) by these lymphocytes and the number of plasmablasts were analyzed by multi-parametric flow cytometry analyses. RESULTS: Increased percentages of Tfh and Tph lymphocytes were detected in patients with HT and GD. Furthermore, an enhanced synthesis of the cytokine IL-21 by these cells was observed. Accordingly, we detected significant higher percentages of plasmablasts in patients with GD, and these values tended to be also higher in HT patients. Moreover, significant positive associations were observed between the levels of Tfh or Tph and the number of plasmablast or anti-TSHR Ab titers in patients with AITD. CONCLUSION: Our data suggest that Tph lymphocytes may have a relevant role in the pathogenesis of AITD.
RESUMO
INTRODUCTION: Growth hormone (GH)-secreting pituitary tumors (GHomas) are the most common acromegaly cause. At diagnosis, most of them are macroadenomas, and up to 56% display cavernous sinus invasion. Biomarker assessment associated with tumor growth and invasion is important to optimize their management. OBJECTIVES: The study aims to identify clinical/hormonal/molecular biomarkers associated with tumor size and invasiveness in GHomas and to analyze the influence of pre-treatment with somatostatin analogs (SSAs) or dopamine agonists (DAs) in key molecular biomarker expression. METHODS: Clinical/analytical/radiological variables were evaluated in 192 patients from the REMAH study (ambispective multicenter post-surgery study of the Spanish Society of Endocrinology and Nutrition). The expression of somatostatin/ghrelin/dopamine system components and key pituitary/proliferation markers was evaluated in GHomas after the first surgery. Univariate/multivariate regression studies were performed to identify association between variables. RESULTS: Eighty percent of patients harbor macroadenomas (63.8% with extrasellar growth). Associations between larger and more invasive GHomas with younger age, visual abnormalities, higher IGF1 levels, extrasellar/suprasellar growth, and/or cavernous sinus invasion were found. Higher GH1 and lower PRL/POMC/CGA/AVPR1B/DRD2T/DRD2L expression levels (P < .05) were associated with tumor invasiveness. Least Absolute Shrinkage and Selection Operator's penalized regression identified combinations of clinical and molecular features with areas under the curve between 0.67 and 0.82. Pre-operative therapy with DA or SSAs did not alter the expression of any of the markers analyzed except for DRD1/AVPR1B (up-regulated with DA) and FSHB/CRHR1 (down-regulated with SSAs). CONCLUSIONS: A specific combination of clinical/analytical/molecular variables was found to be associated with tumor invasiveness and growth capacity in GHomas. Pre-treatment with first-line drugs for acromegaly did not significantly modify the expression of the most relevant biomarkers in our association model. These findings provide valuable insights for risk stratification and personalized management of GHomas.
Assuntos
Acromegalia , Adenoma , Adenoma Hipofisário Secretor de Hormônio do Crescimento , Invasividade Neoplásica , Humanos , Masculino , Feminino , Acromegalia/metabolismo , Pessoa de Meia-Idade , Adulto , Adenoma Hipofisário Secretor de Hormônio do Crescimento/patologia , Adenoma Hipofisário Secretor de Hormônio do Crescimento/metabolismo , Adenoma/metabolismo , Adenoma/patologia , Idoso , Agonistas de Dopamina/uso terapêutico , Biomarcadores Tumorais/metabolismo , Somatostatina/análogos & derivados , Somatostatina/uso terapêutico , Hormônio do Crescimento Humano/metabolismoRESUMO
Although predictors of response to first-generation somatostatin receptor ligands (fg-SRLs), and to a lesser extent to pasireotide, have been studied in acromegaly for many years, their use is still not recommended in clinical guidelines. Is there insufficient evidence to use them? Numerous biomarkers including various clinical, functional, radiological and molecular markers have been identified. The first ones are applicable pre-surgery, while the molecular predictors are utilized for patients not cured after surgery. In this regard, factors predicting a good response to fg-SRLs are specifically: low basal GH, a low GH nadir in the acute octreotide test, T2 MRI hypointensity, a densely granulated pattern, high immunohistochemistry staining for somatostatin receptor 2 (SSTR2), and E-cadherin. However, there is still a lack of consensus regarding which of these biomarkers is more useful or how to integrate them into clinical practice. With classical statistical methods, it is complex to define reliable and generalizable cut-off values for a single biomarker. The potential solution to the limitations of traditional methods involves combining systems biology with artificial intelligence, which is currently providing answers to such long-standing questions that may eventually be finally included into the clinical guidelines and make personalized medicine a reality. The aim of this review is to describe the current knowledge of the main fg-SRLs and pasireotide response predictors, discuss their current usefulness, and point to future directions in the research of this field.
Assuntos
Acromegalia , Receptores de Somatostatina , Somatostatina , Humanos , Receptores de Somatostatina/metabolismo , Receptores de Somatostatina/agonistas , Acromegalia/tratamento farmacológico , Somatostatina/análogos & derivados , Somatostatina/uso terapêutico , Ligantes , Octreotida/uso terapêutico , Hormônio do Crescimento Humano/metabolismo , Biomarcadores/sangue , Resultado do TratamentoRESUMO
Pituitary neuroendocrine tumors (PitNETs) account for approximately 15% of all intracranial neoplasms. Although they usually appear to be benign, some tumors display worse behavior, displaying rapid growth, invasion, refractoriness to treatment, and recurrence. Increasing evidence supports the role of primary cilia (PC) in regulating cancer development. Here, we showed that PC are significantly increased in PitNETs and are associated with increased tumor invasion and recurrence. Serial electron micrographs of PITNETs demonstrated different ciliation phenotypes (dot-like versus normal-like cilia) that represented PC at different stages of ciliogenesis. Molecular findings demonstrated that 123 ciliary-associated genes (eg, doublecortin domain containing protein 2, Sintaxin-3, and centriolar coiled-coil protein 110) were dysregulated in PitNETs, representing the upregulation of markers at different stages of intracellular ciliogenesis. Our results demonstrate, for the first time, that ciliogenesis is increased in PitNETs, suggesting that this process might be used as a potential target for therapy in the future.
Assuntos
Biomarcadores Tumorais , Cílios , Tumores Neuroendócrinos , Neoplasias Hipofisárias , Humanos , Cílios/patologia , Cílios/ultraestrutura , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Neoplasias Hipofisárias/patologia , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/genética , Feminino , Masculino , Tumores Neuroendócrinos/patologia , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/genética , Pessoa de Meia-Idade , Adulto , Idoso , Recidiva Local de Neoplasia/patologia , Invasividade Neoplásica , Imuno-HistoquímicaRESUMO
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the posttranscriptional level. They are emerging as potential biomarkers and as therapeutic targets for several diseases including autoimmune thyroid diseases (AITD). They control a wide range of biological phenomena, including immune activation, apoptosis, differentiation and development, proliferation and metabolism. This function makes miRNAs attractive as disease biomarker candidates or even as therapeutic agents. Because of their stability and reproducibility circulating miRNAs have been an interesting area of research in many diseases, and studies describing their role in the immune response and in autoimmune diseases have progressively developed. The mechanisms underlying AITD remain elusive. AITD pathogenesis is characterized by a multifactorial interplay based on the synergy between susceptibility genes and environmental stimulation, together with epigenetic modulation. Understanding the regulatory role of miRNAs could lead to identify potential susceptibility pathways, diagnostic biomarkers and therapeutic targets for this disease. Herein we update our present knowledge on the role of microRNAs in AITD and discuss on their importance as possible diagnostic and prognostic biomarkers in the most prevalent AITDs: Hashimoto's thyroiditis (HT), Graves' disease (GD) and Graves' Ophthalmopathy (GO). This review provides an overview of the state of the art in the pathological roles of microRNAs as well as in possible novel miRNA-based therapeutic approaches in AITD.
Assuntos
Doenças Autoimunes , Doença de Graves , Oftalmopatia de Graves , Doença de Hashimoto , MicroRNAs , Doenças da Glândula Tireoide , Humanos , MicroRNAs/genética , Reprodutibilidade dos Testes , Predisposição Genética para Doença , Doença de Hashimoto/diagnóstico , Doença de Hashimoto/genética , Doença de Graves/diagnóstico , Doença de Graves/genética , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/genética , Doenças Autoimunes/terapia , Biomarcadores , Doenças da Glândula Tireoide/diagnóstico , Doenças da Glândula Tireoide/genética , Doenças da Glândula Tireoide/terapiaRESUMO
A state of chronic inflammation is common in organs affected by autoimmune disorders, such as autoimmune thyroid diseases (AITD). Epithelial cells, such as thyroid follicular cells (TFCs), can experience a total or partial transition to a mesenchymal phenotype under these conditions. One of the major cytokines involved in this phenomenon is transforming growth factor beta (TGF-ß), which, at the initial stages of autoimmune disorders, plays an immunosuppressive role. However, at chronic stages, TGF- ß contributes to fibrosis and/or transition to mesenchymal phenotypes. The importance of primary cilia (PC) has grown in recent decades as they have been shown to play a key role in cell signaling and maintaining cell structure and function as mechanoreceptors. Deficiencies of PC can trigger epithelial-mesenchymal transition (EMT) and exacerbate autoimmune diseases. A set of EMT markers (E-cadherin, vimentin, α-SMA, and fibronectin) were evaluated in thyroid tissues from AITD patients and controls through RT-qPCR, immunohistochemistry (IHC), and western blot (WB). We established an in vitro TGF-ß-stimulation assay in a human thyroid cell line to assess EMT and PC disruption. EMT markers were evaluated in this model using RT-qPCR and WB, and PC was evaluated with a time-course immunofluorescence assay. We found an increased expression of the mesenchymal markers α-SMA and fibronectin in TFCs in the thyroid glands of AITD patients. Furthermore, E-cadherin expression was maintained in these patients compared to the controls. The TGF-ß-stimulation assay showed an increase in EMT markers, including vimentin, α-SMA, and fibronectin in thyroid cells, as well as a disruption of PC. The TFCs from the AITD patients experienced a partial transition to a mesenchymal phenotype, preserving epithelial characteristics associated with a disruption in PC, which might contribute to AITD pathogenesis.
Assuntos
Doenças Autoimunes , Doença de Hashimoto , Humanos , Transição Epitelial-Mesenquimal , Fibronectinas/metabolismo , Vimentina/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Caderinas/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Background: Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are heterogeneous rare diseases causing malnutrition and cachexia in which the study of body composition may have an impact in prognosis. Aim: Evaluation of muscle and fat tissues by computed tomography (CT) at the level of the third lumbar (L3 level) at diagnosis and at the end of follow-up in GET-NET patients and their relationships with clinical and biochemical variables as predictors of survival. Methodology: Ninety-eight GEP-NET patients were included. Clinical and biochemical parameters were evaluated. Total body, subcutaneous, visceral and total fat areas and very low-density, low-density, normal density, high-density, very high-density and total muscle areas were obtained from CT images. Results: Body composition measures and overall mortality correlated with age, ECOG (Eastern Cooperative Oncology Group performance status) metastases, lactate dehydrogenase (LDH), albumin and urea levels. Although there was no relationship between body composition variables at diagnosis and overall and specific mortality, an increase in low-density muscle and a decrease in normal-density muscle during follow-up were independently correlated to overall (p <0.05) and tumor-cause mortality (p < 0.05). Conclusion: Although body composition measures obtained by CT at diagnosis did not impact survival of GEP-NET patients, a loss of good quality muscle during follow-up was associated with an increased overall and tumor-related mortality. Nutritional status should therefore be supervised by nutrition specialists and an increase in good quality muscle could improve prognosis.
RESUMO
Few studies have considered immune-mediated inflammatory disorders (IMID) together, which is necessary to adequately understand them given they share common mechanisms. Our goal was to investigate the expression of vasoactive intestinal peptide (VIP) and its receptors VPAC1 and VPAC2 in selected IMID, analyze the effect of biological therapies on them, and identify miRNA signatures associated with their expression. Serum VIP levels and mRNA of VPAC and miRNA expression in peripheral blood mononuclear cells were analyzed from 52 patients with psoriasis, rheumatoid arthritis, Graves' disease, or spondyloarthritis and from 38 healthy subjects. IMID patients showed higher levels of VIP and increased expression of VPAC2 compared to controls (p < 0.0001 and p < 0.0192, respectively). Receiver operating characteristic curve analysis showed that the levels of VIP or VPAC2 expression were adequate discriminators capable of identifying IMID. Treatment of IMID patients with anti-TNFα and anti-IL12/23 significantly affected serum VIP levels. We identified miRNA signatures associated with levels of serum VIP and VPAC2 expression, which correlated with IMID diagnosis of the patients. The results indicate that the expression of VIP/VPAC2 is able of identify IMIDs and open up a line of research based on the association between the VIP/VPAC axis and miRNA signatures in immune-mediated diseases.
Assuntos
Artrite Reumatoide , MicroRNAs , Artrite Reumatoide/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , MicroRNAs/genética , RNA Mensageiro , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/genética , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Peptídeo Intestinal Vasoativo/genética , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
CONTEXT: Histone deacetylases (HDACs) and histone acetyltransferases (HAT) have an important role in the regulation of gene transcription as well as in the development and function of CD4+Foxp3+ T regulatory (Treg) cells. Our group and others have reported that patients with autoimmune thyroid disease (AITD) show abnormalities in the levels and function of different Treg cell subsets. OBJECTIVE: We aimed to analyze the levels of expression of several HDACs and the Tip60 HAT in the thyroid gland and immune cells from patients with AITD. METHODS: The expression of HDAC1-11 and the Tip60 HAT, at RNA and protein levels, were determined in thyroid tissue from 20 patients with AITD and 10 healthy controls and these findings were correlated with clinical data. HDAC9 and Tip60 levels were also analyzed in thyroid cell cultures, stimulated or not with proinflammatory cytokines, as well as in different cell subsets from peripheral blood mononuclear cells. RESULTS: Altered expression of different HDACs was observed in thyroid tissue from AITD patients, including a significant increase in HDAC9, at RNA and protein levels. Likewise, HDAC9 expression was increased in peripheral blood mononuclear cells particularly in Treg cells in patients with AITD. In contrast, Tip60 expression was reduced in thyroid gland samples from patients with Hashimoto thyroiditis. CONCLUSION: Our results indicate that HDAC expression is dysregulated in thyroid gland and immune cells from patients with AITD, suggesting involvement in the pathogenesis of this condition.
Assuntos
Doenças Autoimunes/patologia , Biomarcadores/análise , Doença de Hashimoto/patologia , Histona Desacetilases/metabolismo , Adulto , Idoso , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Estudos de Casos e Controles , Citocinas/metabolismo , Feminino , Seguimentos , Doença de Hashimoto/genética , Doença de Hashimoto/imunologia , Doença de Hashimoto/metabolismo , Histona Desacetilases/genética , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Subpopulações de Linfócitos T/imunologiaRESUMO
Cancer cells develop mechanisms that increase nutrient uptake, including key nutrient carriers, such as amino acid transporter 1 (LAT-1) and glucose transporter 1 (GLUT-1), regulated by the oxygen-sensing Von Hippel Lindau-hypoxia-inducible factor (VHL-HIF) transcriptional pathway. We aimed to analyze these metabolic players in gastroenteropancreatic neuroendocrine tumors (GEP-NET) and correlate them with tumor malignancy and progression. LAT-1, GLUT-1, and pVHL expression was analyzed in 116 GEP-NETs and 48 peritumoral tissue samples by immunohistochemistry. LAT-1 was stably silenced using specific shRNA in the human NET BON cell line. LAT-1 expression was significantly increased in tumor tissue compared to non-tumor tissue in both gastrointestinal (67% vs. 44%) and pancreatic NETs (54% vs. 31%). Similarly, GLUT-1 was substantially elevated in gastrointestinal (74% vs. 19%) and pancreatic (58% vs. 4%) NETs. In contrast, pVHL expression was decreased (85% vs. 58%) in pancreatic NETs. Tumors with metastases at diagnosis displayed increased LAT-1 and GLUT-1 and decreased pVHL expression (p < 0.001). In accordance with these data, silencing LAT-1 curtailed cell proliferation in BON cells. These findings suggest that specific mechanisms that increase nutrient uptake, such as LAT-1 and GLUT-1, are increased in GEP-NETs, whereas pVHL is decreased. These markers might be related to the proliferation and metastatic capacity of these tumors.
RESUMO
CONTEXT: Natural killer (NK) cells have an important role in innate immunity and in the regulation of immune response. The role of NK cells expressing the programmed cell death protein-1 (PD-1) regulatory receptor has not been explored in patients with autoimmune thyroid disease (AITD). PURPOSE: To analyze the levels and function of PD-1+ NK cells in samples from AITD patients. DESIGN: Cases and controls, observational study. SETTING: Hospital Universitario la Princesa, Spain. PATIENTS: Forty patients with AITD, 16 with Hashimoto thyroiditis (HT), 24 with Graves' disease (GD), and 15 healthy controls. INTERVENTION: Multiparametric flow cytometry analysis of peripheral blood NK cells. In vitro assays of cytotoxic activity of NK cells, and synthesis of cytokines. MAIN OUTCOME MEASURES: Levels and function of PD-1+ NK cells in blood samples from AITD patients and controls. RESULTS: Increased levels of NK cells and the CD56dimPD-1+ subset were observed in GD patients. In HT, an enhanced expression of the regulatory receptors NKG2A and NKG2C by CD56brightPD-1+ NK cells was detected. AITD patients showed an increased synthesis of IL-10 by CD56brightPD-1- NK cells, whereas CD56dimPD-1+ cells from GD patients exhibited an enhanced production of interferon-γ. PD-1+ NK cells from patients with GD and HT showed an increased cytotoxic activity. Significant associations were observed in patients with GD or HT between the levels of PD-1+ NK cells and clinical laboratory parameters. CONCLUSIONS: The different abnormalities in NK cell subset levels, in the expression of PD-1 and its function in AITD patients' further support the complex role of these cells in this pathogenesis.
Assuntos
Doença de Graves/imunologia , Doença de Hashimoto/imunologia , Células Matadoras Naturais/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Adulto , Feminino , Citometria de Fluxo , Doença de Graves/sangue , Doença de Hashimoto/sangue , Humanos , Imunidade Inata , Masculino , Pessoa de Meia-IdadeRESUMO
Immune-mediated inflammatory disorders (IMID) are a group of diseases that present inflammation as a major pathogenic mechanism. They affect 15% of the population and pose a heavy socio-economic burden. Despite the growing knowledge on the etiopathogenesis of these diseases and the marked improvement in their management, there is a lack of predictive markers of IMID development or severity suitable for early diagnosis and adjustment of treatment intensity. The possibility that certain circulating miRNA profiles could be used as biomarkers of risk of development and/or severity of several autoimmune diseases has fuelled the interest in using them to improve the selection of successful treatments. The multi-pronged approach proposed here sought to reveal circulating miRNAs and miRNA signatures that could act as new predictive biomarkers of IMID development and severity. Our results showed that the circulating levels of miR-19b and miR-26b were significantly decreased (p < 0.001) in IMID patients compared to controls. Furthermore, receiver operating characteristic (ROC) curve analysis showed that these miRNAs were suitable discriminators capable to identify an IMID, with areas under the curve (AUC) of 0.85 and 0.83, respectively. In addition, we established that miR-19a and miR-143 were significantly increased in IMID patients with severe disease (p < 0.05). In summary, our findings identify two different miRNA signatures. One of them is associated with the presence of IMIDs and could lead to the development of tools for their early detection. The second signature is able to discriminate between mild and severe forms of these disorders and could be a putative tool to select patient candidates for a more intense treatment.
Assuntos
Doenças Autoimunes/diagnóstico , MicroRNA Circulante/genética , Inflamação/diagnóstico , MicroRNAs/genética , Adulto , Doenças Autoimunes/genética , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica , Marcadores Genéticos , Humanos , Inflamação/genética , Masculino , Pessoa de Meia-Idade , Risco , Índice de Gravidade de Doença , TranscriptomaAssuntos
Doença de Hashimoto , Tireoidite Autoimune , Humanos , Linfócitos , Linfócitos T ReguladoresRESUMO
BACKGROUND: The mechanisms underlying autoimmune thyroid disease (AITD) remain elusive. Identification of such mechanisms would reveal novel and/or better therapeutic targets. Here, we use integrated analysis of miRNAs and mRNAs expression profiling to identify potential therapeutic targets involved in the mechanisms underlying AITD. METHODS: miRNA and mRNA from twenty fresh-frozen thyroid tissues (15 from AITD patients and 5 from healthy controls) were subjected to next-generation sequencing. An anti-correlated method revealed potential pathways and disease targets, including proteins involved in the formation of primary cilia. Thus, we examined the distribution and length of primary cilia in thyroid tissues from AITD and controls using immunofluorescence and scanning electron microscopy, and parsed cilia formation in thyroid cell lines in response to inflammatory stimuli in the presence of miRNA mimics. FINDINGS: We found that the expression of miR-21-5p, miR-146b-3p, miR-5571-3p and miR-6503-3p was anti-correlated with Enolase 4 (ENO4), in-turned planar cell polarity protein (INTU), kinesin family member 27 (KIF27), parkin co-regulated (PACRG) and serine/threonine kinase 36 (STK36) genes. Functional classification of these miRNA/mRNAs revealed that their differential expression was associated with cilia organization. We demonstrated that the number and length of primary cilia in thyroid tissues was significantly lower in AITD than in control (frequency of follicular ciliated cells in controlsâ¯=â¯67.54% vs a mean of 22.74% and 21.61% in HT and GD respectively p = 0.0001, by one-way ANOVA test). In addition, pro-inflammatory cytokines (IFNγ and TNFα) and specific miRNA mimics for the newly identified target genes affected cilia appearance in thyroid cell lines. INTERPRETATION: Integrated miRNA/gene expression analysis has identified abnormal ciliogenesis as a novel susceptibility pathway that is involved in the pathogenesis of AITD. These results reflect that ciliogenesis plays a relevant role in AITD, and opens research pathways to design therapeutic targets in AITD. FUNDING: Instituto de Salud Carlos III, Comunidad de Madrid, Grupo Español de Tumores Neuroendocrinos y Endocrinos, Ministerio de Economía y Empresa and FEDER.
Assuntos
Doenças Autoimunes/etiologia , Estudos de Associação Genética , Predisposição Genética para Doença , MicroRNAs/genética , RNA Mensageiro/genética , Doenças da Glândula Tireoide/etiologia , Adulto , Doenças Autoimunes/diagnóstico , Autoimunidade , Biomarcadores , Biópsia , Biologia Computacional/métodos , Citocinas/metabolismo , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Imuno-Histoquímica , Mediadores da Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Doenças da Glândula Tireoide/diagnósticoRESUMO
The immune checkpoint based therapy targeting the programmed death-1 (PD-1) receptor and its PD-L1 ligand has recently been approved for the therapy of different malignant conditions, but not yet for gastroenteropancreatic neuroendocrine tumors (GEP-NETs). In this context, we evaluated the expression of PD-1 and PD-L1 in GEP-NETs and its potential correlations with clinical outcomes. Expression of PD-1/PD-L1 was analyzed by immunohistochemistry in 116 GEP-NETs and 48 samples of peritumoral tissue. In addition, the expression of these molecules was assessed by flow cytometry in peripheral blood mononuclear cells (PBMC) from patients with GEP-NETs (n = 32) and healthy controls (n = 32) and in intratumoral mononuclear cells (TMCs) (n = 3). Expression of PD-L1 and PD-1 was detected by immunohistochemistry in 6% and 1% of tumor tissue samples, respectively, and in 8% of peritumoral tissue samples, for both markers. We also observed that PD-1 expression by TMCs was associated with metastatic disease at diagnosis, and the levels of circulating PD-1+ PBMCs were associated with progressive disease upon follow-ups. In addition, circulating PD-1+ PBMCs were significantly correlated with PD-L1 expression by tumor cells. Our data suggest that PD-1/PD-L1 is expressed in 1 to 8% of GEP-NETs, and that this feature is significantly associated with disease evolution (p < 0.01).
Assuntos
Antígeno B7-H1/biossíntese , Regulação Neoplásica da Expressão Gênica , Neoplasias Intestinais , Proteínas de Neoplasias/biossíntese , Tumores Neuroendócrinos , Neoplasias Pancreáticas , Receptor de Morte Celular Programada 1/biossíntese , Neoplasias Gástricas , Adulto , Idoso , Feminino , Humanos , Neoplasias Intestinais/diagnóstico , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/patologia , Masculino , Pessoa de Meia-Idade , Tumores Neuroendócrinos/diagnóstico , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Prognóstico , Estudos Retrospectivos , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologiaRESUMO
Context: T regulatory type 1 (Tr1) cells are a subpopulation of T lymphocytes (CD4+CD49+LAG-3+IL-10+) that exert a considerable immunosuppressive effect. However, their possible role in autoimmune thyroid disease (AITD) has not been explored so far. Purpose: To analyze the levels and function of Tr1 cells in peripheral blood and thyroid tissue of patients with AITD. Design: Cases and controls, observational study. Setting: Department of Endocrinology, Hospital Universitario de la Princesa, Madrid, Spain. Patients: Thirty-eight patients with AITD (23 with Graves disease and 15 with Hashimoto thyroiditis) and 26 controls. Intervention: Multiparametric flow cytometry and immunofluorescence techniques were used to analyze the levels in peripheral blood (n = 38) and thyroid mononuclear cells (n = 5). An in vitro assay of suppression of cellular activation and cytokine release was performed to study the function of Tr1 cells. Main Outcome Measure: Levels and function of Tr1 cells in patients with AITD and controls. Results: Levels of Tr1 cells were significantly diminished in peripheral blood from patients with AITD. Functional studies showed that Tr1 cells from patients with AITD exhibit a diminished suppressive function compared with healthy controls. Tr1 levels were associated with disease severity, including longer duration of the disease and ophthalmopathy activity, and with autoantibody titers. Conclusions: The low levels of Tr1 cells and their diminished function may have a relevant role in the defective immune-regulatory function characteristic of patients with AITD.
Assuntos
Doença de Graves/sangue , Doença de Hashimoto/sangue , Ativação Linfocitária/imunologia , Linfócitos T Reguladores/imunologia , Tireoidite Autoimune/sangue , Adulto , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Imunofluorescência , Doença de Graves/imunologia , Doença de Hashimoto/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Glândula Tireoide/imunologia , Tireoidite Autoimune/imunologiaRESUMO
Context: Circulating microRNAs (miRNAs) are emerging as an interesting research area because of their potential role as novel biomarkers and therapeutic targets. Their involvement in autoimmune thyroid diseases (AITDs) has not been fully explored. Objective: To compare the expression profile of miRNAs in thyroid tissue from patients with AITD and controls, using next-generation sequencing, further validated our findings in thyroid and serum samples. Design: Twenty fresh-frozen thyroid tissues (15 from patients with AITD and 5 from controls) were used for miRNA next-generation sequencing. Thirty-six thyroid samples were recruited for the qRT-PCR validation test and 58 serum samples for further validation in peripheral blood. Results: Expression of several miRNAs that had been previously associated with relevant immunological functions was significantly dysregulated. Specifically, eight differentially expressed miRNAs (miR-21-5p, miR-142-3p, miR-146a-5p, miR-146b-5p, miR-155-5p, miR-338-5p, miR-342-5p, and miR-766-3p) were confirmed using qRT-PCR in thyroid samples, and three had the same behavior in tissue and serum samples (miR-21-5p, miR-142-3p, and miR-146a-5p). Furthermore, when the expression of these miRNAs was assessed together with five additional ones previously related to AITD in peripheral blood, the expression of five (miR-Let7d-5p, miR-21-5p, miR-96-5p, miR-142-3p, and miR-301a-3p) was significantly expressed in AITD and, in patients with Graves disease (GD), was correlated with a higher severity of disease, including active ophthalmopathy, goiter, higher antibody titers, and/or higher recurrence rates. Conclusions: The present findings identify a serum five-signature miRNA that could be an independent risk factor for developing AITD and a predisposition of a worse clinical picture in patients with GD.
Assuntos
MicroRNAs/análise , Índice de Gravidade de Doença , Glândula Tireoide/metabolismo , Tireoidite Autoimune/genética , Adulto , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Doença de Graves/genética , Doença de Graves/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Risco , Glândula Tireoide/patologia , Tireoidite Autoimune/patologiaRESUMO
PURPOSE: To study the levels of pathogenic and non-pathogenic Th17 and Th22 cells in autoimmune thyroid disorders patients. Although Th17 cells seem to play an important role in the pathogenesis of thyroid autoimmune disorders, the specific subsets of these lymphocytes have not been analyzed in this condition. METHODS: We assessed the levels of Th17 (pathogenic and non-pathogenic) and Th22 cells in peripheral blood and thyroid glands of autoimmune thyroid disorders patients (n = 26, 16 with Graves' disease and 10 with Hashimoto's thyroiditis) and 15 healthy controls by multi-parametric flow cytometry and immunofluorescence microscopy. RESULTS: We found increased levels of pathogenic Th17 lymphocytes and Th22 cells in peripheral blood from autoimmune thyroid disorders patients. In addition, these cells were detected in thyroid glands from HT patients. Furthermore, we found significant correlations between the levels of these cells and disease activity, disease duration, and the presence of ophthalmopathy. CONCLUSIONS: The increased levels of pathogenic Th17 lymphocytes and Th22 cells in autoimmune thyroid disorders suggest their involvement in the pathogenesis of this condition.
Assuntos
Autoimunidade , Doença de Graves/imunologia , Doença de Hashimoto/imunologia , Linfocitose/etiologia , Linfócitos T Auxiliares-Indutores/imunologia , Células Th17/imunologia , Glândula Tireoide/imunologia , Adulto , Autoanticorpos/análise , Células Cultivadas , Feminino , Citometria de Fluxo , Doença de Graves/metabolismo , Doença de Graves/patologia , Doença de Graves/fisiopatologia , Oftalmopatia de Graves/etiologia , Doença de Hashimoto/metabolismo , Doença de Hashimoto/patologia , Doença de Hashimoto/fisiopatologia , Humanos , Iodo/urina , Ativação Linfocitária , Contagem de Linfócitos , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/patologia , Células Th17/metabolismo , Células Th17/patologia , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Glândula Tireoide/fisiopatologia , Hormônios Tireóideos/sangueRESUMO
Context: Signaling lymphocytic activation molecule family 1 (SLAMF1) is a costimulatory receptor expressed by most immune cells. Its role in autoimmune thyroid disease (AITD) is not well known. Objective: To analyze the expression and function of the costimulatory receptor SLAMF1 in lymphocytes of patients with AITD. Design: Cross-sectional, prospective, single-center study. Setting: Department of Endocrinology, Hospital Universitario de la Princesa, Madrid. Patients: Twenty-eight patients with AITD (17 with Graves disease and 11 with Hashimoto thyroiditis) and 21 controls. Intervention: Multiparametric flow cytometry and immunofluorescence techniques to analyze the expression of SLAMF1 in peripheral blood (n = 28) and thyroid tissue (n = 5) mononuclear cells. Assay of inhibition of cellular proliferation to study the function of SLAMF1 in CD4+CD25+ T regulatory (Treg) cells. Main Outcome Measure: Expression levels and the function of SLAMF1 in lymphocytes in AITD patients and controls. Results: Expression of SLAMF1 was significantly increased in peripheral blood CD4+, T helper 17, and CD19+ B cells from AITD patients. Immunofluorescence microscopy detected the presence of SLAMF1+ lymphocytes in thyroid inflammatory cell infiltrate. Functional studies showed that SLAMF1 engagement in Treg cells increased their suppressive function in healthy controls but not in AITD patients. Conclusions: The altered expression of SLAMF1, as well as its defective function observed in patients with AITD, may have a relevant role in the defective immune-regulatory function observed in this condition.